Article Assessing the Pathogenicity of Two Isolated from the Entomopathogenic indica against and Some Pest Insects

Rosalba Salgado-Morales 1,2 , Fernando Martínez-Ocampo 2 , Verónica Obregón-Barboza 2, Kathia Vilchis-Martínez 3, Alfredo Jiménez-Pérez 3 and Edgar Dantán-González 2,*

1 Doctorado en Ciencias, Instituto de Investigación en Ciencias Básicas y Aplicadas, Universidad Autónoma del Estado de Morelos, Av. Universidad 1001, Chamilpa, 62209 Cuernavaca, Morelos, Mexico; [email protected] 2 Laboratorio de Estudios Ecogenómicos, Centro de Investigación en Biotecnología, Universidad Autónoma del Estado de Morelos, Av. Universidad 1001, Chamilpa, 62209 Cuernavaca, Morelos, Mexico; [email protected] (F.M.-O.); [email protected] (V.O.-B.) 3 Centro de Desarrollo de Productos Bióticos, Instituto Politécnico Nacional, Calle Ceprobi No. 8, San Isidro, Yautepec, 62739 Morelos, Mexico; [email protected] (K.V.-M.); [email protected] (A.J.-P.) * Correspondence: [email protected]; Tel.: +52-777-329-7000

 Received: 20 December 2018; Accepted: 15 March 2019; Published: 26 March 2019 

Abstract: The entomopathogenic Heterorhabditis are parasites of insects and are associated with mutualist symbiosis enterobacteria of the genus ; these bacteria are lethal to their host insects. Heterorhabditis indica MOR03 was isolated from sugarcane soil in Morelos state, Mexico. The molecular identification of the nematode was confirmed using sequences of the ITS1-5.8S-ITS2 region and the D2/D3 expansion segment of the 28S rRNA gene. In addition, two bacteria HIM3 and NA04 strains were isolated from the entomopathogenic nematode. The genomes of both bacteria were sequenced and assembled de novo. Phylogenetic analysis was confirmed by concatenated gene sequence datasets as HIM3 (16S rRNA, 23S rRNA, dnaN, gyrA, and gyrB genes) and Pseudomonas aeruginosa NA04 (16S rRNA, 23S rRNA and gyrB genes). H. indica MOR03 infects Galleria mellonella, Tenebrio molitor, Heliothis subflexa, and magnifactella larvae with LC50 values of 1.4, 23.5, 13.7, and 21.7 IJs/cm2, respectively, at 48 h. These bacteria are pathogenic to various insects and have high injectable insecticide activity at 24 h.

Keywords: entomopathogenic nematode; Heterorhabditis indica; bacteria; pathogenicity; insects

1. Introduction The entomopathogenic nematodes (Rhabditida: Heterorhabditidae and Steinernematidae) are parasites of insects that are associated in mutualist symbiosis with enterobacteria of the genera Photorhabdus and Xenorhabdus [1,2]. The entomopathogenic nematodes, unlike other parasites, cause the death of the host in a short time (within 24–48 h of infection), and this lethality is provided by associated bacteria [2]. The infective juvenile stage (IJ) is the only free life form of the entomopathogenic nematodes and functions as a vector carrying the symbiotic bacteria in the intestine to release them in the host insect’s hemolymph [3]. The life cycle of the nematode is completed inside the insect cadaver. The symbiotic bacteria of entomopathogenic nematodes (Photorhabdus and Xenorhabdus) are , Gram-negative, and members of the family Enterobacteriaceae. Canonically,

Insects 2019, 10, 83; doi:10.3390/insects10030083 www.mdpi.com/journal/insects Insects 2019, 10, 83 2 of 14

Photorhabdus and Xenorhabdus establish monoxenic associations with Heterorhabditis and Steinernema, respectively. They are virulent to a wide variety of insects including Coleoptera, Hemiptera, , and Diptera. The mean lethal dose (LD50) is low: <100 colony forming unit (CFU) injected into the hemolymph of Galleria mellonella larvae L (Lepidoptera: Pyralidae) [4–8]. The diversity of the toxins produced, lipasses, and proteases, as well as the ability of these bacteria to suppress the insect’s immune system, contribute to its high virulence. In addition, the secondary metabolites produced by the bacteria are essential for the development of the nematode [9]. However, non-canonical bacteria such as Providencia sp., Ochrobactrum sp., Pseudomonas sp., and Alcaligenes faecalis have been isolated from different entomopathogenic nematodes, and few studies have focused on the role of these bacteria and their association with the nematode [10–13]. Due to the lifestyle of entomopathogenic nematodes, it is interesting to evaluate the pathogenic activity of non-canonical bacteria in insects. Particularly, Heterorhabditis indica has been reported in diaxenic association with Photorhabdus luminescens and Ochrobactrum spp., and the latter does not have insecticidal activity against G. mellonella. In contrast, A. faecalis from Heterorhabditis sp. is pathogenic to G. mellonella, suggesting a possible role in pathogenesis in insects [11–13]. G. mellonella is widely used as a model insect to evaluate bacterial pathogenesis and virulence [14,15]. The larvae of G. mellonella have technical advantages such as that they are easy to maintain under laboratory conditions, no special equipment is needed, and virulence bioassays by injection can be made using larvae of the last instar. Subsequently, bacterial virulence can be determined by calculating the LD50 [16,17]. Particularly, G. mellonella has been used extensively for the study of the pathogenicity of entomopathogenic nematodes and their symbiotic bacteria [18]. However, there is great interest in evaluating the pathogenicity of entomopathogenic nematodes and their symbiotic bacteria (canonical and non-canonical) in other insects, mainly in insects considered pests of agricultural crops. In this study, we report the isolation, identification, and pathogenicity of the entomopathogenic nematode Heterorhabditis indica MOR03 and its associated bacteria (canonical and non-canonical) in larvae of the G. mellonella model and larvae of pest insects: Tenebrio molitor L. (Coleoptera: Tenebrionidae), Heliothis subflexa Dyar (Lepidoptera: Noctuidae), and Diatraea magnifactella Dyar (Lepidoptera: ).

2. Materials and Methods

2.1. Isolation of the Entomopathogenic Nematodes A total of 23 soil samples were collected in agricultural soils with a history of pesticide application of Morelos, Mexico, in the months of February to June 2014. Approximately 1 kg of soil was taken at a depth of 10 to 30 cm and the isolation of entomopathogenic nematodes was carried out using G. mellonella last-instar larvae as bait [19]. In the laboratory, the larvae were monitored daily, and the dead larvae were removed every day. Dead larvae with symptoms of infection by entomopatogenic nematodes such as absence of odor, absence of contaminating organisms, and coloration were recovered and disinfected with 5% sodium hypochlorite and rinsed 3 times with sterile distilled water before being transferred to White traps where nematodes were obtained from the insect larvae [20]. Koch postulates were performed to confirm that the nematodes were the cause of death [21].

2.2. Maintenance of Insects and Nematodes The insects (G. mellonella, H. subflexa, and D. magnifactella) were kept at a temperature of 27 ± 2 ◦C with a light/darkness (LD) photoperiod of 12:12 h, kept at relative humidity (RH) 70 ± 10%, and grown on artificial diets. Only T. molitor was maintained at room temperature. The nematodes were reproduced into last-instar G. mellonella larvae and the IJs were recovered from the White traps after 12–14 days of inoculation with IJs. Insects 2019, 10, 83 3 of 14

2.3. Molecular Identification of the Entomopathogenic Nematodes Genomic DNA (gDNA) was extracted from IJs using the DNA Isolation Kit for Cells and Tissues (Roche, Indianapolis, IN, USA, following the manufacturer’s instructions). The primers TW81 and AB28 were used for amplification of the DNA fragment containing the ITS1-5.8S-ITS2 region of rRNA genes [22]. The D2/D3 expansion segment of the 28S rRNA gene was amplified using the primers D2F and 536 [23,24]. PCR products were purified using a QIAquick Gel Extraction kit® (Qiagen Inc., Santa Clara, CA, USA) and sequenced in both directions using the same primers at the Unidad de Síntesis y Secuenciación de DNA (USSD) of the Instituto de Biotecnología (IBT)-UNAM. The sequence reads were assembled and edited using BioEdit Version 7.2.5 software [25]. The resulting sequences were aligned using the Nucleotide BLAST tool NCBI GenBank (https://blast.ncbi.nlm.nih. gov/Blast.cgi). For phylogenetic reconstruction, we downloaded 25 ITS1-5.8S-ITS2 sequences and 19 D2/D3 sequences of the expansion segment of the 28S rRNA of species in the genera Heterorhabditis and Caenorhabditis. Multiple alignments between generated sequences and downloaded sequences selected from the GenBank database were made using the MUSCLE (version 3.8.31) program [26]. The jModelTest (version 2.1.10) [27] was used to select the model of nucleotide substitution using the Akaike information criterion (AIC). Phylogenetic trees were inferred based on maximum likelihood for both datasets using the PhyML (version 3.1) program [28] with 1000 bootstrap replicates. Phylogenetic trees were visualized and edited using the FigTree (version 1.4.3) program (http://tree.bio.ed.ac.uk/ software/figtree/).

2.4. Determination of the LC50 of the Nematode against Insects Bioassays to determine the mean lethal concentration, LC50, in G. mellonella and T. molitor consisted of using 10 and 15 larvae per dose tested, which were confined in 9-cm Petri dishes floored with Whatman no. 2 filter paper disks. Each treatment had five doses: 0.7, 1.5, 3.1, 6.2, 12.5, and 15.7 and 7.8, 15.7, 31.4, 62.8, and 157.2 IJs/cm2, respectively. The doses used to determine the LC50 for H. subflexa and D. magnifactella were 4, 8, 16, 32, and 64 and 4, 8, 16, 24, and 48 IJs/cm2, respectively. These last ones were confined individually in a 6-well plate Costar (Corning Life, New York, NY, USA). The concentrations of nematodes were adjusted by volumetric dilutions in a final volume of 1 mL according to the methodology described by Glazer and Lewis, 2000 [29]. In all cases, sterile distilled water was used as a negative control and larvae were incubated at 28 ± 2 ◦C. Mortality was evaluated 48 h after inoculation and at least three independent bioassays were conducted. Mortality data was analyzed using Probit (version 1.0) to determine the LC50 with PoloPlus software [30].

2.5. Bacteria Isolation from the Nematodes Bacteria were isolated from the hemolymph of infected larvae with isolate H. indica MOR03. Disinfection of the larvae of G. mellonella was performed using 2% (w/v) benzalkonium chloride and rinsing with sterile distilled water before allocation into 9-cm Petri dishes. Subsequently, 1.5 IJs/cm2 (previously disinfected) were added. After 38 h of infection, the larvae were disinfected under sterile conditions and the hemolymph was obtained using insulin syringe (BD Medical-Diabetes Care, Holdrege, NE, USA) with a 31-gauge needle; the hemolymph droplet was inoculated on the nutrient bromothymol blue agar (NBTA) and incubated at 28 ± 2 ◦C for 72 h. To obtain a fresh culture of bacteria from the IJs, approximately 1000 IJs were disinfected with 0.1% (w/v) benzalkonium chloride solution for 20 min, then rinsed with sterile distilled water and macerated with a Teflon micropestle under sterile conditions. A 10-µL sample of the macerated material was inoculated in Luria–Bertani (LB) broth and NBTA medium and incubated at 28 ± 2 ◦C for 24 h [12]. The experiment was carried out three times using different generations of IJs of H. indica MOR03 and the identification of the bacteria was confirmed by sequencing of the 16S rRNA gene. Insects 2019, 10, 83 4 of 14

2.6. Sequencing, Assembly, and Genome Annotation The gDNA of HIM3 and NA04 strains was extracted using the ZR Fungal/Bacterial Kit MiniPrepTM (Zymo Research, Irvine, CA, USA, following the manufacturer’s instructions), and 5 µg of gDNA was sequenced with the Illumina HiSeq platform (2 × 300 bp Paired-End reads). Bacterial genomes were assembled de novo using the SPAdes (version 3.5.0) program [31] and automated annotation of bacterial genomes was performed using the RAST (version 2.0) server [32]. The annotations resulting from the genomes were used to search for virulence factors based on keywords (pathogenesis, virulence, toxin, adhesion, fimbriae). The prediction of rRNA and tRNA genes was performed using the RNAmmer (version 1.2) [33] and ARAGORN [34] servers, respectively [35,36].

2.7. Molecular Identification of Bacteria The 16S rRNA, 23S rRNA, dnaN, gyrA, and gyrB sequences, and 16S rRNA, 23S rRNA, and gyrB sequences of housekeeping genes were obtained from the genomes of HIM3 and NA04 strains, respectively. The gene sequence datasets from the HIM3 and NA04 genomes were compared to six downloaded gene sequence datasets of the genera Photorhabdus and Proteus, and 16 downloaded gene sequence datasets of the genera Pseudomonas and Escherichia, which were obtained from the GenBank database (https://www.ncbi.nlm.nih.gov/) using the nucleotide BLAST suite (https://blast.ncbi.nlm. nih.gov/Blast.cgi). Multiple alignments between gene sequence datasets of the HIM3 and NA04 strains and downloaded gene sequence datasets selected from the GenBank database were made using the MUSCLE (version 3.8.31) program [26]. Alignment sequences per genome were concatenated using a Python script. The jModelTest (version 2.1.10) [27] was used to select the model of nucleotide substitution using the Akaike information criterion (AIC). Phylogenetic trees were inferred based on maximum likelihood for each concatenated gene dataset using the PhyML (version 3.1) program [28] with 1000 bootstrap replicates. Phylogenetic trees were visualized and edited using the FigTree (version 1.4.3) program (http://tree.bio.ed.ac.uk/software/figtree/).

2.8. Bacterial Pathogenicity against Insects The bacterial isolates of the hemolymph (HIM3), the macerate of IJs (NA04), and E. coli DH5α (negative control) were individually inoculated in 100 mL of LB liquid medium and grown overnight at 30 ◦C and 150 rpm. Viable counts were made, and the doses were adjusted in a final volume of 10 µL. Each bacterial suspension was injected individually into an insect’s hemolymph at the last left pro-leg into G. mellonella, T. molitor, and D. magnifactella larvae by using insulin syringe (BD Medical-Diabetes Care, Holdrege, NE, USA) with a 31-gauge needle. LB medium, puncture, and equivalent doses of E. coli DH5α were used as negative controls. Approximately, the doses used on G. mellonella were 10–120 CFU HIM3 and 25–400 CFU NA04, and for T. molitor, 1000–10,000 CFU HIM3 and 400–5000 CFU NA04 were used. Mortality was determined 24 h after injection. D. magnifactella mortality was evaluated 24 and 36 h after injection at 3000 and 10,000 CFU of HIM3 and NA04, respectively. Fifteen larvae were used per dose and each larva was placed in a 9-cm Petri dish with 0.5 g of artificial diet and incubated at 28 ± 2 ◦C. The experiment was repeated in three independent bioassays and the data were analyzed using Probit version 1.0 to determine the LD50 with PoloPlus software [30].

3. Results

3.1. Isolation and Molecular Identification of H. indica A single isolate of entomopathogenic nematode was obtained from one sample, corresponding to 4.3% of 23 samples. The positive sample was collected in a field cultivated with sugarcane in the town of Oacalco in Yautepec, Morelos, Mexico (18◦55016.9” N and 99◦02024.0” W). The infection characteristics present in the larvae of G. mellonella infected with entomopathogenic nematode were Insects 2019, 10, 83 5 of 14 no odor or copper red coloration. The IJs emerged from the infected larvae between 12 and 14 days after infection. The ITS1-5.8S-ITS2 sequence of our isolate MOR03 is 99% similar to H. indica (strain D1, GenBank accession number AY170329). Additionally, the 28S D2/D3 expansion segment sequence is 99% similar toInsectsH. indica 2019, 10(strain, FOR PEER Cohen REVIEW 21, GenBank accession number GU177842). Two maximum likelihood trees5 15 (Figure1a,b) show that the entomopathogenic nematode isolated belongs to the H. indica species. Theindica model species. of nucleotide The model substitution of nucleotide (selected substitution by jModelTest) (selected wasby jModelTest) TMV+G, for was both TMV+G, ITS1-5.8S-ITS2 for both andITS1-5.8S-ITS2 28S D2/D3 and datasets. 28S D2/D3 datasets.

(a)

Figure 1. Cont.

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(b)

FigureFigure 1. 1.Phylogenetic Phylogenetic relationshipsrelationships basedbased onon thethe regions regions of of rRNA rRNA gene gene sequences sequences of ofHeterorhabditis Heterorhabditis indicaindica MOR03MOR03 (blue letters) with with 24 24 and and 17 17 sequences sequences of ofgenus genus HeterorhabditisHeterorhabditis, respectively., respectively. (a) (aPhylogenetic) Phylogenetic tree tree of the of theITS1-5.8S-ITS2 ITS1-5.8S-ITS2 region region of rRNA of rRNA gene gene sequences. sequences. (b) Phylogenetic (b) Phylogenetic tree of tree the ofD2/D3 the D2/D3 expansion expansion segment segment of 28S of rRNA 28S rRNA gene genesequence sequences.s. Phylogenetic Phylogenetic trees trees were were obtained obtained using using the thePhyML PhyML (version (version 3.1) 3.1) program program with with the the maximum maximum likelihood likelihood method method and and 1000 1000 bootstrap replicates.replicates. BootstrapBootstrap values values are are displayed displayed as as a a percentage percentage of of confidence confidence (>70%) (>70%) in in the the nodes. nodes.

3.2.3.2. PathogenicityPathogenicity andand VirulenceVirulence of of H. H. indica indica MOR03 MOR03 to to Insects Insects TheThe isolatedisolatedH. H. indicaindicaMOR03 MOR03 isisa apathogen pathogento toseveral severalLepidoptera— Lepidoptera—G.G. mellonellamellonella,, H.H. subflexasubflexa,, andandD. D. magnifactella magnifactella—and—and the the coleopteran coleopteranT. molitorT. molitorin laboratory in laboratory conditions. conditions. The values The values of LC50 of showLC50 thatshow the that order the of order virulence of virulence of H. indica of H.MOR03 indica isMOR03G. mellonella is G. mellonella> H. subflexa > H.> subflexaD. magnifactella > D. magnifactella> T. molitor > (TableT. molitor1). The (Table dose–response 1). The dose–response curves are curves shown are in Figure shown S1 in ofFigure the Supplementary S1 of the Supplementary Materials. Materials.

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Table 1. LC50 (IJs/cm2) values of Heterorhabditis indica MOR03 against G. mellonella, T. molitor, Table 1. LC50 (IJs/cm2) values of Heterorhabditis indica MOR03 against G. mellonella, T. molitor, H. H. subflexa, and D. magnifactella obtained at 48 h. subflexa, and D. magnifactella obtained at 48 h. 2 2 Insect Insect n n InstarInstar LC50LC50 (IJs/cm (IJs/cm) 95%2) 95% CI CI χχ2 df df G. mellonellaG. mellonella320 320 Last Last instar instar 1.4 1.4 (1.0–1.9) (1.0–1.9) 33.8 33.8 30 30 T. molitorT. molitor 210210 15th15th instar instar 23.5 23.5 (15.8–33.3) (15.8–33.3) 6.9 6.9 12 12 H. subflexa 225 Last instar 13.7 (10.1–18.3) 12.3 13 D. magnifactellaH. subflexa 204225 5thLast instar instar 21.7 13.7 (14.4–43.8) (10.1–18.3) 7.712.3 13 15 D. magnifactella 204 5th instar 21.7 (14.4–43.8) 7.7 15 Larvae were incubated at 28 ± 2 ◦C in darkness. (p < 0.05). Larvae were incubated at 28 ± 2 °C in darkness. (p < 0.05).

3.3.3.3. IsolationIsolation ofof Bacteria,Bacteria, GenomeGenome Sequencing,Sequencing, and and Molecular Molecular Identification Identification TwoTwo bacterial bacterial isolates isolates were were obtained; obtained; HIM3 HIM3 was was isolated isolated from from the the hemolymph hemolymph of of larvae larvae infected infected withwithH. H. indica indicaMOR03; MOR03; HIM3 HIM3 and NA04and NA04 were isolatedwere isolated from the from maceration the maceration of IJs in different of IJs generations.in different Ingenerations. addition, the In bacterialaddition, genomes the bacterial of the genomes HIM3 and of the NA04 HIM3 strains and wereNA04 sequenced strains were and sequenced assembled and de novo.assembled The draft de novo. genomes The draft of HIM3 genomes and NA04 of HIM3 strains and haveNA04 a strains total length have ofa total 5,472,253 length bp of in 5,472,253 91 contigs bp within 91 a contigs G+C content with a G+C of 42% content and aof total 42% lengthand a tota of 6,375,895l length of bp 6,375,895 in 54 contigs bp in 54 with contigs a G+C with content a G+C ofcontent 66.4%, of respectively. 66.4%, respectively. Both genome Both genome sequences sequences were deposited were deposited in the GenBankin the GenBank database database under accessionunder accession numbers numbers GCA_002204205.1 GCA_002204205.1 and GCA_002204155.1, and GCA_002204155.1, respectively respectively [35,36]. The [35,36]. automated The annotationautomated ofannotation the bacterial of the genomes bacterial shows genomes that theshows HIM3 that strain the HIM3 contains strain 4659 contains coding 4659 sequences coding (CDS),sequences 70 tRNAs, (CDS), 70 and tRNAs, 10 rRNAs; and 10 the rRNAs; NA04 the strain NA contains04 strain 5823contains CDS, 5823 67 tRNAs,CDS, 67 andtRNAs, 7 rRNAs. and 7 MaximumrRNAs. Maximum likelihood likelihood trees of trees five of (16S five rRNA (16S rRNA and 23Sand rRNA,23S rRNA,dnaN dnaN,, gyrA gyrA, and, andgyrB gyrB) and) and three three (16S(16S rRNA,rRNA, 23S rRNA, rRNA, and and gyrBgyrB) concatenated) concatenated gene gene sequence sequence data datasetssets show show that thatthe HIM3 the HIM3 and NA04 and NA04strains strains are grouped are grouped with with the thePhotorhabdusPhotorhabdus luminescens luminescens andand PseudomonasPseudomonas aeruginosa species,species, respectivelyrespectively (Figure(Figure2 a,b).2a,b). The The HIM3 HIM3 strain strain is is closely closely related related to toPhotorhabdus Photorhabdus luminescensluminescensDSM DSM3368 3368 (GenBank(GenBank accession accession number number NZ_JXSK00000000.1) NZ_JXSK00000000.1) isolated isolated from fromH. bacteriophora H. bacteriophorain Australia; in Australia; the NA04 the strainNA04 is strain related is related to Pseudomonas to Pseudomonas aeruginosa aeruginosaM18 (GenBank M18 (GenBank accession accession number number CP002496) CP002496) isolated isolated from rhizospherefrom rhizosphere soil of sweetsoil of melon sweet [37 melon]. Therefore, [37]. Therefore, we named we these named bacteria thesePhotorhabdus bacteria Photorhabdus luminescens HIM3luminescens and Pseudomonas HIM3 and Pseudomonas aeruginosa NA04. aeruginosa NA04.

(a)

Figure 2. Cont.

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(b)

FigureFigure 2.2. PhylogeneticPhylogenetic relationshipsrelationships basedbased onon thethe concatenatedconcatenated genegene sequencesequence datasetsdatasets ofof thethe twotwo bacteriabacteria strainsstrains HIM3HIM3 andand NA04NA04 isolatedisolated fromfrom thethe entomopathogenicentomopathogenic nematodenematode H.H. indicaindica str.str. MOR03.MOR03. (a(a)) PhylogeneticPhylogenetic tree based on on the the concatenated concatenated sequ sequencesences of offive five housekeeping housekeeping genes genes (16S (16S rRNA rRNA and and23S 23SrRNA, rRNA, dnaN,dnaN gyrA, gyrA, and, andgyrBgyrB) of) Photorhabdus of Photorhabdus luminescens luminescens str. str.HIM3 HIM3 (blue (blue letters) letters) with with five fiveconcatenated concatenated gene gene sequence sequence datasets datasets of ofgenus genus PhotorhabdusPhotorhabdus. .((bb) )Phylogenetic Phylogenetic tree tree basedbased onon concatenatedconcatenated sequences sequences of of three three housekeeping housekeeping genes genes (16S (16S rRNA, rRNA, 23S 23S rRNA, rRNA, and andgyrB gyrB)) of ofPseudomonas Pseudomonas aeruginosaaeruginosastr. str. NA04 NA04 (blue(blue letters)letters) withwith 1515 concatenatedconcatenated genegene sequencesequence datasetsdatasets ofof genusgenus Pseudomonas.Pseudomonas. PhylogeneticPhylogenetic trees trees were were obtained obtained using using the PhyML the PhyML (version (version 3.1) program 3.1) program with the maximumwith the likelihoodmaximum methodlikelihood and method 1000 bootstrap and 1000 replicates. bootstrapBootstrap replicates. values Bootstrap are displayed values are as displayed a percentage as a ofpercentage confidence of (>70%)confidence in the (>70%) nodes. in the nodes.

TheThe genomegenome of P.of luminescensP. luminescensHIM3 HIM3 contains contains CDS that CDS code that forproteins code for involved proteins in pathogenesis,involved in suchpathogenesis, as effector such proteins as effector (VirK pr andoteins YopT), (VirK lipoproteins and YopT), involved lipoproteins in adhesion involved and in adhesion colonization and colonization (NlpE, ShlA, galactophilic lectin PA-I), fimbrial adhesion precursors, siderophore production and iron-regulated proteins, proteins involved in the evasion of the immune system

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(NlpE,(palmitoyltransferase ShlA, galactophilic pagP), lectin hemolysins, PA-I), fimbrial and adhesiona large number precursors, of insecticidal siderophore toxins. production On the andother iron-regulatedhand, the genome proteins, of proteinsP. aeruginosa involved NA04 in theeffector evasion proteins of the (YopR, immune HopPmaj) system (palmitoyltransferase includes exotoxin A pagP),precursor, hemolysins, accessory and cholera a large enterotoxin, number of and insecticidal zona occludens toxins. toxin, On the as otherwell as hand, hemolysin, the genome adhesion, of P.and aeruginosa siderophoreNA04 effectorproduction proteins proteins. (YopR, Both HopPmaj) HIM3 and includes NA04 exotoxinstrains contain A precursor, genes that accessory code for cholera toxins enterotoxin,of the RTX andfamily zona (Tables occludens S1,S2, toxin, supplementary as well as hemolysin,materials). adhesion, and siderophore production proteins. Both HIM3 and NA04 strains contain genes that code for toxins of the RTX family (Tables S1 and3.4. S2, Pathogenicity Supplementary of P. luminescens Materials). HIM3 and P. aeruginosa NA04 in Insects The bacteria P. luminescens HIM3 and P. aeruginosa NA04 are pathogenic to the larvae of G. 3.4. Pathogenicity of P. luminescens HIM3 and P. aeruginosa NA04 in Insects mellonella and cause a high percentage of mortality at 24 h. The LD50 value of P. luminescens HIM3 to G. Themellonella bacteria is 19P. (14.4–23.8 luminescens 95%HIM3 CI) CFU/larvae, and P. aeruginosa χ2 = 16.1.NA04 Similarly, are pathogenic when 25 toto the400 larvaeCFU of of P. G.aeruginosa mellonella NA04and cause were ainjected high percentage into G. mellonella of mortality larvae, at almost 24 h. 100% The LD50 mortality value rates of P. were luminescens observed HIM3(Figure to G. 3). mellonella These resultsis 19 (14.4–23.8 indicate 95%that CI) both CFU/larvae, HIM3 andχ 2NA04= 16.1. isolates Similarly, are when highly 25 tovirulent 400 CFU to of G. P.mellonella aeruginosa. TheNA04 bioassays were injected with intoT. molitorG. mellonella showedlarvae, that a almost high dose 100% (1491 mortality CFU/larva, rates were χ2 = observed 7.9) of P. (Figureaeruginosa3). These NA04 results is required indicate to that cause both 50% HIM3 mortality and NA04 at 24 isolates h and arethat highly doses virulent (1000–10,000 to G. mellonellaCFU) of .P. Theluminescens bioassays HIM3 with T.cause molitor betweenshowed 20% that and a high 40%dose mortality (1491 in CFU/larva, T. molitor χat2 =24 7.9) h. ofInterestingly,P. aeruginosa no NA04mortality is required was observed to cause at 50% 24 h mortality when 3000 at 24and h 10,000 and that CFU doses of HIM3 (1000–10,000 and NA04 CFU) were of injectedP. luminescens into the HIM3hemolymph cause between of D. magnifactella 20% and 40%. However, mortality at 36 in h,T. approximately molitor at 24 h. 100% Interestingly, mortality nowas mortality observed was with observedHIM3 and at 24only h when 10% mortality 3000 and 10,000with NA04 CFU (Figure of HIM3 4). and NA04 were injected into the hemolymph of D. magnifactella. However, at 36 h, approximately 100% mortality was observed with HIM3 and only 10% mortality with NA04 (Figure4).

100

75

50

25 Mean (+SEM) mortality (%)

0 LB P Ec 25 50 100 200 400

CFU FigureFigure 3. 3.Mean Mean ( ±(± SEM)SEM) mortality of GalleriaGalleria mellonella mellonella afterafter inoculation inoculation with with P.P. aeruginosa aeruginosa NA04.NA04. LB LB= =Luria Luria–Bertani–Bertani broth, broth, P P= =puncture, puncture, Ec Ec = =E.E. coli coli DH5DH5α α(25,(25, 50, 50, 100, 100, 200, 200, and and 400 400 CFU). CFU).

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100

90

24 H 36 H 80 20 Mean (+SEM) Mean mortality (%) 10

0 LB PEcHIM3NA04EcHIM3NA04 FigureFigure 4. 4.Mean Mean ( ±(± SEM)SEM) mortality of D.D. magnifactella magnifactella atat 24 24 and and 36 36 h hafter after inoculation inoculation with with HIM3 HIM3 and andNA04 NA04 at at3000 3000 (empty (empty bars) bars) and and 10,000 10,000 CFU CFU (solid (solid bars bars),), respectively. respectively. Error Error bars bars are arethe theSEM. SEM. LB = LBLuria = Luria–Bertani–Bertani broth, broth P ,= P puncture, = puncture, Ec Ec = =EschericiaEschericia coli coli (3000(3000 and and 10,00010,000 CFU)CFU) HIM3 HIM3 = =P. P. luminescens luminescens HIM3,HIM3, NA04 NA04 = P.= P. aeruginosa aeruginosaNA04. NA04. 4. Discussion 4. Discussion Heterorhabditis indica’s preference is for regions with warm climates, including tropical and Heterorhabditis indica’s preference is for regions with warm climates, including tropical and subtropical regions in North and South America, the Caribbean, Africa, Asia, and Australia [38]. subtropical regions in North and South America, the Caribbean, Africa, Asia, and Australia [38]. The The entomophatogenic nematode H. indica MOR03 was isolated from agricultural soil cultivated with entomophatogenic nematode H. indica MOR03 was isolated from agricultural soil cultivated with sugarcane in the town of Oacalco in Yautepec, Morelos, where the average temperature is around sugarcane in the town of Oacalco in Yautepec, Morelos, where the average temperature is around 22.7 ◦C and the climate is warm and subhumid. H. indica’s preference for warm climates suggests that 22.7 °C and the climate is warm and subhumid. H. indica’s preference for warm climates suggests that it possesses advantageous characteristics to inhabit this geographic zone; this may include adaptation it possesses advantageous characteristics to inhabit this geographic zone; this may include adaptation to high temperatures, tolerance to desiccation, and good dispersibility [39]. to high temperatures, tolerance to desiccation, and good dispersibility [39]. The main phylogenetic analyses of the genus Heterorhabditis are based on the partial 18S, 28S, The main phylogenetic analyses of the genus Heterorhabditis are based on the partial 18S, 28S, ITS1, and ITS1-5.8-ITS2 regions of rRNAs genes. Nevertheless, the analysis of the D2/D3 expansion ITS1, and ITS1-5.8-ITS2 regions of rRNAs genes. Nevertheless, the analysis of the D2/D3 expansion segment of the 28S rRNA gene resolved the relationship between Heterorhabditis species. Actually, segment of the 28S rRNA gene resolved the relationship between Heterorhabditis species. Actually, only one genome of genus Heterorhabditis has been reported in the GenBank database: the draft only one genome of genus Heterorhabditis has been reported in the GenBank database: the draft genome of H. bacteriophora M31e (GenBank accession number ACKM00000000.1). Therefore, there is genome of H. bacteriophora M31e (GenBank accession number ACKM00000000.1). Therefore, there is not enough information available to perform a phylogenetic analysis of concatenated housekeeping not enough information available to perform a phylogenetic analysis of concatenated housekeeping genes or the core genome. In this work, we selected two different regions to identify the species of the genes or the core genome. In this work, we selected two different regions to identify the species of entomopathogenic nematode isolated: ITS1-5.8S-ITS2 and the D2/D3 expansion segment of rRNA the entomopathogenic nematode isolated: ITS1-5.8S-ITS2 and the D2/D3 expansion segment of rRNA genes. Also, we used a character-based method for phylogenetic reconstruction, the maximum genes. Also, we used a character-based method for phylogenetic reconstruction, the maximum likelihood (ML) method, which is a probabilistic method and requires the selection of a model likelihood (ML) method, which is a probabilistic method and requires the selection of a model of of nucleotide substitution from 88 models available. We highlight the importance of sequencing nucleotide substitution from 88 models available. We highlight the importance of sequencing the the genomes of the genus Heterorhabditis to reliably study the evolutionary history, geographical genomes of the genus Heterorhabditis to reliably study the evolutionary history, geographical distribution and ecology of this genus. distribution and ecology of this genus. H. indica MOR03 is a pathogen in all tested insects but with different degrees of virulence H. indica MOR03 is a pathogen in all tested insects but with different degrees of virulence depending on the host insect. According to other reports, the LC50 varies with respect to the nematode depending on the host insect. According to other reports, the LC50 varies with respect to the species, its virulence, and the host insect. For example, [40] reported that 25 (10.2 IJs/cm2) and nematode species, its virulence, and the host insect. For example, [40] reported that 25 (10.2 IJs/cm2) 55 IJs (20.5 IJs/cm2) are required to achieve 50% mortality of G. mellonella with H. indica, but almost and 55 IJs (20.5 IJs/cm2) are required to achieve 50% mortality of G. mellonella with H. indica, but three times more H. bacteriophora, 160 IJs, (66.6 IJs/cm2) are needed to achieve the same mortality. almost three times more H. bacteriophora, 160 IJs, (66.6 IJs/cm2) are needed to achieve the same Our data show that H. indica MOR03 is more virulent than this, as only 1.4 IJs/cm2 were required to

Insects 2019, 10, 83 11 of 14 kill 50% of the G. mellonela larvae. A similar LC50, 1.99–6.9 IJs per G. mellonella larva (0.9–3.4 IJs/cm2), was reported for the four isolates of H. indica [18]. The values of LC50 that we obtained for T. molitor, H. subflexa, and D. magnifactella are 9 to 16 times larger than that for G. mellonella. This confirms that G. mellonella is the most susceptible host insect to H. indica MOR03 and is consistent with other reports [18]. The LC50 values of H. indica MOR03 on insects of economic interest as H. subflexa and D. magnifactella (<25 IJs/cm2) show its potential as a biological control agent [41]. However, field studies are required for validation. In this work, we evaluated the pathogenicity and virulence of two bacteria isolated from H. indica MOR03 against insects. An interesting aspect is that, in addition to P. luminescens HIM3, the non-canonical bacterium P. aeruginosa NA04 exhibited entomopathogenic activity in some insects, but not in D. magnifactella. Both bacteria are pathogens and highly virulent (almost 100% mortality after 24 h) against G. mellonella. A 100% mortality rate after 24 h was reported when injecting the same dose of P. aeruginosa CFU into G. mellonella [42]. However, susceptibility to HIM3 and NA04 is affected by the host insect. T. molitor required doses in excess of 1000 CFU. However, D. magnifactella was affected by P. luminescens HIM3 and not by P. aeruginosa NA04. This suggests that P. luminescens HIM3 has a larger arsenal of insecticidal toxins or greater specificity for D. magnifactella. Interestingly, the non-canonical bacterium P. aeruginosa NA04 was not found in the hemolymph of G. mellonella larvae infected with H. indica MOR03 at 36 h after infection. However, it is possible that the non-canonical bacterium P. aeruginosa NA04 is released or reproduces in the hemolymph in later hours. Furthermore, the presence of non-canonical bacteria in JIs from different generations suggests that the bacteria are retained by H. indica MOR03. The non-canonical bacteria P. aeruginosa NA04 could be contributing to the pathogenesis of the nematode only in some insects (G. mellonella and T. mollitor) in laboratory conditions. In contrast, P. luminescens HIM3 is pathogenic for all test insects. Both bacteria contain in their genome the genes that code for the lectins involved in the process of colonization and invasion of the host, such as galactophilic lectin PA-I and proteins for the synthesis of fimbria, another important factor for colonization [43,44]. The genome of P. luminescens HIM3 presents the gene coding for a palmitoyltransferase (pagP), which modifies the lipid A of lipopolysaccharide (LPS); this modification allows the bacteria to inhibit the inflammatory response and evade the insect immune response [45]. The PhoP–PhoQ two-component system is associated with virulence in P. luminescens and P. aeruginosa, and it is contained in the genome of HIM3 and NA04 [46–48]. The acquisition of iron in pathogenic bacteria is essential to overcome the immune response of the host. The strains HIM3 and NA04 have a variety of genes for the production of siderophores, small iron binding molecules that are secreted by bacteria to acquire ferric iron the host [49]. In particular, P. luminescens HIM3 contains large proteins with possible insecticidal activity as well as proteins that frame the type III secretion system (T3SS) responsible for introducing the virulence proteins into the cytoplasm of eukaryotic cells; the differences in the range of effector proteins secreted by each bacterium could be related to its degree of virulence and specificity [50]. In addition to pathogenicity, T3SS plays an important role in symbiotic relationships [51,52]. It should be noted that the presence of the nematode plays an important role in the suppression of the host´s immune system, as the H. bacteriophora cuticle causes host immunosuppression through the inhibition of eicosanoid biosynthesis in G. mellonella [53].

5. Conclusions This paper reports the isolation and molecular identification of the nematode H. indica MOR03 that has proven to be lethal for various insects. The insecticidal activity could be related to the presence of two bacteria (canonical and non-canonical) which were also identified. The relationship of the canonical bacteria of the genus Photorhabdus with entomopathogenic nematodes of the genus Heterorhabditis has been extensively reported. However, there have been few studies focused on the role played by non-canonical bacteria. This report shows that P. aeruginosa NA04 has insecticidal activity for different insects but that this activity is different from that of P. luminescens HIM3, which would help to clarify Insects 2019, 10, 83 12 of 14 how symbiotic bacteria work when the nematode infects different hosts. They would also provide new resources and alternatives for further studies of symbiotic bacteria of the entomopathogenic nematode, for example, the role that bacteria play in the specificity that the nematode has on insects, due to toxins and other mechanisms that contain the associated bacteria. Entomopathogenic nematodes and their associated bacteria (canonical and non-canonical) are an appropriate model to test hypotheses regarding the role that each organism plays in the various activities of the nematode and to understand the complex mechanisms of symbiotic relationships.

Supplementary Materials: The following are available online at http://www.mdpi.com/2075-4450/10/3/83/s1, Figure S1: Dose–response curves and LC50 of H. indica MOR03 against insects; Table S1: Proteins involved in the pathogenesis Photorhabdus luminescens HIM3; Table S2: Proteins involved in the pathogenesis Pseudomonas aeruginosa NA04. Author Contributions: Conceptualization, R.S.-M. and E.D.-G.; methodology, R.S.-M., F.M.-O., V.O.-B. and K.V.-M.; formal analysis, R.S.-M., F.M.-O, A.J.-P. and E.D.-G.; investigation, R.S.-M. and E.D.-G.; resources, E.D.-G.; data curation, R.S.-M. A.J.-P. and E.D.-G.; writing—original draft preparation, R.S.-M., F.M.-O., A.J.-P. and E.D.-G.; writing—review and editing: R.S.-M., A.J.-P. and E.D.-G.; project administration, E.D.-G.; funding acquisition, E.D.-G. Funding: This research was funded by CONACyT scholarships of R.S.-M., numbered 296934, and a project of E.D.-G., with project number CONACyT PDCPN 0247780. Conflicts of Interest: The authors declare no conflict of interest.

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